Field of Invention
The present invention relates generally to detection assays of biomolecular entities, such as nucleotides and proteins, and more specifically, to detection methods and systems that use multiple light emitting molecules having emissions in different colors for use to identify numerous different entities in the assay.
Description of Related Art
Technologies related to the recognition and detection of DNA sequences in solution is the basis of several different analytical assay approaches, which can be used for analysis of different genome DNAs and, in particular, identification of genetically diverse living organisms. The identification of living organisms, detection of different microorganism mutations and strains of pathogenic bacteria, which cause severe diseases in humans, by means of quantitative analysis of their specific DNA sequences is a challenging goal, and is the focus of much research today1-5.
In the last few decades significant progress in DNA analysis has been achieved by the discovery and implementation of the PCR approach for the analysis of genetic material6,7. PCR is a hypersensitive method by which a few fragments of DNA can be duplicated into millions in a couple of hours. In other words, it represents a DNA copying machine based on an artificial increase in the amount of DNA, containing the specific target sequence. After amplification, DNA material can be easily detected by common analytical methods. Despite the obvious advantage of PCR in DNA detection this approach has some disadvantages2,8,9, e.g. sensitivity to DNA material contaminants, misreading, quite high cost of analysis, reagents and time to fulfill experiments, and most importantly, limited utility as a general fast and easy Point-of-Care method of specific DNA sequence quantification2,3.
Another approach for DNA quantitation is based on the direct detection of a small amount of DNA in solution, i.e. without any amplification of the DNA material. It is based on detection of the bright emission of dyes bound to nucleic acids10-12. Most popular chromophores for this approach are ethidium bromide, PicoGreen and Syber Green I, which bind DNA non-covalently and subsequently increase their fluorescence yield. For example, the last two chromophores increase their brightness almost 1,000 fold upon binding to double stranded DNA10,11,13-15. It makes them extremely sensitive to a small (<ng/ml) amounts of DNA in solution. Moreover, it recently has been shown that in the presence of silver nanoparticles, due to the Metal-Enhanced Fluorescence (MEF) effect16, the sensitivity of PicoGreen and Syber Green I to dsDNA can be significantly further increased and become comparable to the sensitivity of the PCR technique, i.e. to be in the range of ˜pg/ml11. The significant benefit of this approach is both the speed and the inexpensive nature of DNA quantitation. Disadvantages of this approach include a lack of DNA sequence specificity, which makes it unfeasible to employ directly in analysis of genome specific DNA samples.
A remarkable improvement of this technique17,18 has been achieved by the combination of two approaches: microwave accelerated sequence-specific hybridization of the target DNA with anchor DNA, immobilized on a metal surface, and the Metal-Enhanced Fluorescence (MEF) effect, responsible for the immense enhancement of a DNA's fluorescent label. The MEF effect, i.e. enhancement of a fluorophore's brightness, exponentially depends on the distance between a chromophore and metallic nanoparticle, due to a short-range (0-30 nm) coupling of a chromophore's excited state electronic system with nanoparticle (NP) plasmons. As a result, only chromophores proximal to NPs increase their emission hundred's-thousand fold. Subsequently, hybridization is not only the event of a specific recognition of a target DNA, but also the creation of the MEF pair (fluorophore—NP plasmons), which enhances the fluorescence signal. Duplex annealing puts a fluorescent label on a short (˜7 nm) enough leash, relative to a NP, thereby placing the label in the perfect condition for intense MEF16,22,23. A significant addition to this technology is microwave “heating” of the reacting system, which significantly speeds up the process of DNA hybridization24, which is an important attractive feature of any bio-assay.
Notably the above systems are limited to locating one entity at a time, thereby increasing time and cost to separate and identify more that one target nucleotide or protein in the assay. As such, numerous assays need to be completed to identify separated targets. Thus, it would be advantageous to have systems and methods that have the ability to locate numerous targets at the same time while exhibiting increased efficiency and reaction time.